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EcoCRM TM

A Recombinant CRM 197 Carrier Protein

A. Lees 1 , R. Simon 2 , S. Baliban 2 , I. Krauss 3 , D. Nguyen 3 , M. Pravetoni 4,5 , N. Oganesyan 1

1, 2 Fina Biosolutions, Rockville, MD; 2 U Maryland School of Med, Baltimore; 3 Brandeis U, Waltham, MA; 4 Minneapolis Medical Research Foundation (MMRF); 5 Center for Immunology, U Minnesota

EcoCRM TM Affordable CRM 197 Carrier Protein CRM 197 , a genetically detoxified diphtheria toxin,
EcoCRM TM
Affordable CRM 197 Carrier Protein
CRM 197 , a genetically detoxified diphtheria toxin, is widely used as a carrier protein
in conjugate vaccines. It was originally expressed as a secreted protein in
Corynebacterium diphtheriae . Until recently, both “native” and recombinant CRM 197
has been difficult to obtain and/or expensive.
Fina BioSolutions has developed a new CRM 197 , EcoCRM TM , produced in E.coli .
EcoCRM TM is expressed as soluble protein in the cytoplasm of a widely-used E.coli
expression strain. High expression of EcoCRM TM in a BL21 strain and a simple
purification method allows low cost production and the promise of significantly
reducing the cost of this component of conjugate vaccines.
The data presented show that EcoCRM TM is an excellent carrier protein for a
variety of antigens.
SDS PAGE EcoCRM TM
>99% purity. No nicking evident.
SEC HPLC of EcoCRM TM
Dimer is <0.6%. Purity >99%.
Therapeutic Vaccines for Treatment of
Opioid Abuse and Overdose
The Pravetoni lab has developed a series of conjugate vaccines that elicit opioid-specific IgG
antibodies that reduce opioid distribution to the brain. Immunization selectively blocks opioid-
induced behaviors, such as opioid self-administration, and prevents opioid-induced respiratory
depression and bradycardia in mice and rats. Current efforts focus on readying for FDA approval
and Phase I clinical trial a vaccine consisting of oxycodone (OXY) conjugated to a GMP
monomer KLH carrier protein. Due to its mullosk origin and poor characterization, native KLH is
not an ideal carrier. This study compared oxycodone conjugates using EcoCRM TM and KLH.
The haptenization ratio of
oxycodone-EcoCRM TM can
be characterized by MALDI-
TOF.
EcoCRM TM was analyzed before and
after conjugation to the OXY hapten. A
ratio of 24 haptens per CRM was
calculated from the MW difference. We
were unable to characterize the
conjugates containing native KLH.
IN VIVO VACCINE EFFICACY
A
B
400
Induction of oxycodone-specific IgG antibody
prevents oxycodone distribution to the brain
in mice .
1000
**
800
300
***
(58%)
600
****
200
400
(73%)
****
BALB/c mice (n ≥ 4/group) were immunized s.c. with 100 µg
unconjugated KLH (control), OXY-KLH, or OXY-EcoCRM TM ,
formulated with 1 mg of alum adjuvant on days 0, 14 and 28. On
day 35, mice were challenged with 2.25 mg/kg oxycodone, and
then brain and serum collected for analysis of vaccine efficacy.
100
200
A) Oxycodone-specific serum IgG titers analyzed by ELISA.
0
0
B) Oxycodone concentration in the brain 30-min after oxycodone
challenge, analyzed by GC/MS. Above bars, percentages (%)
indicate decrease in brain oxycodone compared to KLH.
C
D
0.0020
**
0.0060
**
Induction of oxycodone-specific B cells
0.0015
0.0045
**
0.0010
0.0030
0.0005
0.0015
BALB/c mice (n ≥ 4/group) were immunized i.m. with 100 µg
KLH, OXY-KLH, or OXY-EcoCRM TM , formulated with 200 µg of
alum. At 7 days post-immunization, OXY-specific B cell
populations from spleen and lymph nodes were isolated by
antigen-based magnetic enrichment paired with multi-parameter
flow cytometry.
0.0000
0.0000
C) Oxycodone-specific IgM + B cells.
D) Oxycodone-specific germinal center (GC) B cells.
The oxycodone-EcoCRM TM conjugate showed superior efficacy than the previously
established oxycodone-KLH. In contrast to KLH conjugates, EcoCRM TM conjugates are
KLH
An oxycodone-EcoCRM TM
conjugate demonstrated similar or superior
OXY-KLH
easier to characterize.
efficacy against oxycodone compared to the standard KLH carrier protein.
OXY-EcoCRM TM
The Pravetoni group is currently developing vaccines and monoclonal antibodies
against heroin, oxycodone, and fentanyl abuse and overdose (Pubmed, “Pravetoni M”).
For collaborations or partnership, please contact
Dr. Marco Pravetoni at prave001@umn.edu.
KLH
KLH
OXY-KLH
OXY-KLH
OXY-EcoCRM TM
OXY-EcoCRM TM
KLH
OXY-KLH
OXY-EcoCRM TM
Oxycodone-specific IgM + B cells
(% of lymphocytes)
Oxycodone-specific IgG titer (x10 3 )
OXY-specific GC B cells
(% of lymphocytes)
Brain oxycodone (ng/mL)

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www.PosterPresentations.com

S. Typhimurium Core-OPS-EcoCRM TM Conjugate Development of a glycoconjugate vaccine to prevent invasive Salmonella
S. Typhimurium Core-OPS-EcoCRM TM Conjugate
Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium
infections in sub-Saharan Africa. Baliban et al. PLoS Negl Trop Dis 11(4):e0005493, 2017
STm-COPS was covalently linked
to EcoCRM TM via its KDO group
S. Typhimurium core O-
polysaccharide (STm-COPS)
STm-COPS KDO -EcoCRM TM
neoglycoconjugate
P
< 0 .0 0 0 1
P
< 0 .0 0 0 1
1 0 0
P
< 0 .0 0 0 1
1 0 7
8
0
P
B
S
(lo w d o se)
1 0 6
6
0
P
B
S
(h ig h
d o se )
1 0 5
1 0 4
P
S -C R M
(lo w do se)
4
0
1 0 3
P
S -C R M
(h ig h
d o se )
2
0
1 0 2
1 0 1
0
1 0 0
0
2
4
6
8
1 0
1 2
1 4
P B S
d o s e )
P B S
P S -C R M
P S -C R M
T im e
p o st-ch a lle n g e
(d a ys)
(lo w
(hig h
d o s e )
(lo w
d o s e )
(hig h
d o s e )
Kaplan-Meier survival curves of mice immunized with
Serum IgG titers against STm-COPS from mice (n =
STm-COPSKDO-CRM
(circle) or PBS (diamond) after
HIV Glycopeptide Conjugate
KDO
197
20/group) immunized with 2.5 µg STm-COPS
:CRM
197
challenge (n = 20/group) with 1 or 5x10 6 CFU of STm D65.
or PBS. Solid bars indicate the GMT.
Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope
S. Typhimurium COPS-EcoCRM
TM
conjugate
Bailey et al. Tetrahedron 72:6092, 12016
provided 100% protection in this animal model
HIV Glycopeptide-EcoCRM TM Conjugate
Synthesis of multivalent glycopeptide conjugates that mimic an HIV epitope.
Bailey et al. Tetrahedron. 72:6091, 2016.
Thio-ether conjugation is frequently used for linking peptides, oligosaccharides
and other antigens to a carrier protein such as CRM 197 .
EcoCRM TM was labeled with an excess of the NHS maleimide
reagent sulfo-EMCS. The MW of EcoCRM TM increased from
58,413 to 62,525, (delta=4100). Each linker adds a mass of 208,
indicating about 20 of CRM’s 39 lysines have been labeled.
Thiol-glycopeptide was conjugated to the
EMCS-labeled CRM 197 . Mass spec of the
conjugates indicated ~7 glycopeptides/CRM.
Glycopeptide conjugates of EcoCRM TM and
CRM from P. fluorescens (“ Pfenex”) were
prepared. Rabbits were immunized twice.
After the final bleed,IgG anti-glycopeptide
titers were measured using glycopeptide-BSA
as the ELISA antigen.
Glycopeptide conjugates with
EcoCRM TM & Pfenex CRM induced
comparable anti-glycopeptide titers
in rabbits.
Pfenex
Fina Biosolutions
www.FinaBio.com
Info@FinaBio.com
EcoCRM TM is an effective carrier protein for
conjugate vaccines and is an alternative to
CRM 197 from Corynebacterium and
Pseudomonas. A simple expression system,
high yields and the efficient purification of
EcoCRM TM can help reduce the cost of
conjugate vaccines.
-P S serum IgGa
(E U /m L)
S urvival (% )